Structure and Genetic Diversity of Anacardium Humile (Anacardiaceae): a Tropical Shrub

Structure and genetic diversity of Anacardium humile (Anacardiaceae): a tropical shrub L.G. Cota1, P.A. Moreira2, M.M. Brandão1, V.A. Royo1, A.F. Melo Junior1, E.V. Menezes1 and D.A. Oliveira1 1Laboratório de Bioprospecção e Recursos Genéticos, Departamento de Biologia Geral, Universidade Estadual de Montes Claros, Campus Universitário Professor Darcy Ribeiro, Montes Claros, MG, Brasil 2Departamento de Biologia, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, Ouro Preto, MG, Brasil Corresponding author: E.V. Menezes E-mail: [email protected] Genet. Mol. Res. 16 (3): gmr16039778 Received July 17, 2017 Accepted August 29, 2017 Published September 27, 2017 DOI http://dx.doi.org/10.4238/gmr16039778 Copyright © 2017 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution ShareAlike (CC BY-SA) 4.0 License. ABSTRACT. Anacardium humile Saint Hilaire is a tropical shrub native to the Cerrado biome. It is a fruiting species with biological, medicinal, and socioeconomic significance. Thus, knowing how the genetic variability of natural populations is organized allows for the establishment of strategies for conservation and the sustainable use of the species and its biome. Six microsatellite loci previously developed from Anacardium occidentale were used to investigate the spatial genetic structure and genetic diversity of eight natural A. humile populations based on analyses of 242 adult plants. The results obtained indicate that these populations show a high level of genetic diversity (expected heterozygosity = 0.710). The endogamy coefficient was positive and significant for most populations, with a mean of 0.142 (P = 0.001). The genetic differentiation between populations was low (θ = 0.075 and GST = 0.066) but significant (P = 0.0001). The genotypes Genetics and Molecular Research 16 (3): gmr16039778 L.G. Cota et al. 2 of five of the eight populations were non-randomly distributed with clusters of related plants for which the coancestry values were positive and significant. These populations exhibited high and significant endogamy indices. The results obtained for A. humile populations show that genetic conservation programs should be implemented to maintain this species. Key words: Cashew of Cerrado; Microsatellite markers; Cerrado; Structure and genetic diversity INTRODUCTION Changes in tropical landscapes resulting from fragmentation accentuate the loss of genetic diversity as they reduce population sizes. When small populations become isolated and thus more susceptible to effects associated with endogamy, genetic drift, and absent and/or limited gene flow (Frankham et al., 2002), and also the capacity of a species to evolve suffers (Hamrick, 2004). Therefore, maintaining genetic diversity is critical, as it is a fundamental component of biological diversity and allows species to adapt to environments through a constant process of transformation (Frankham et al., 2002). In recent years, tropical biomes have been greatly degraded, making their conservation of utmost importance. This is especially true for the Cerrado, the second largest Brazilian biome, which contains great biological diversity and many habitats and is the source of various natural resources (Cavalcanti and Joly, 2002). The Cerrado is considered a global biodiversity hotspot based on the anthropic impacts it has suffered, the number of endemic species it houses, and the high level of species diversity in general (Myers et al., 2000). The Cerrado has suffered great damage from alterations to its landscape due to the expansion of human activities in the region, such as the use of the land for agriculture and ranching, exploitation of the native flora and fauna, and predatory extractivism (Henriques, 2003). These activities have put the biodiversity of Cerrado at risk, and early studies on this topic looked at the accelerated fragmentation of its natural habitats (Machado et al., 2004). Thus, the Cerrado is an interesting region for studying the effects of human occupation on the genetics of plant populations and how genetic variation relates to the characteristics of life history (Collevatti et al., 2010). Numerous studies on the genetic conservation of native Cerrado plant species have been carried out, analyzing these populations using microsatellite molecular markers (Zucchi et al., 2003; Moura et al., 2009; Collevatti et al., 2010; Tarazi et al., 2010). This technique is widely used because it is codominant, highly polymorphic and informative (Tautz, 1989; Guichoux et al., 2011). Interest in genetic studies of natural populations that combine evolutionary biology and ecology has increased in recent years (Frankham et al., 2002). Quantifying the levels of genetic diversity and understanding how populations are genetically structured allow for the identification of populations and priority sites for conservation. Additionally, it is possible to monitor long-term changes in gene flow, endogamy levels, genetic structure, and effective population sizes of species in altered habitats and/or exploited species (Gibbs, 2001; Frankham et al., 2002). These conservation activities integrate genetics with demographic and environmental variables, catastrophes, and human impact to predict extinction risks, and compare alternative options in species recovery programs (Frankham, 2003). Genetics and Molecular Research 16 (3): gmr16039778 Genetic diversity of Anacardium humile 3 This study aims to assess how natural populations of Anacardium humile, an important tropical shrub species native to the Cerrado biome, are genetically structured. The level of genetic diversity was quantified in these populations to obtain information for the conservation, management, and appropriate use of the species and its biome. MATERIAL AND METHODS Species studied A. humile Saint Hilaire is a tropical fruiting species in the Anacardiaceae family. The species is commonly known as cashew of Cerrado because it is native to rocky savannas in the Cerrado biome (Almeida et al., 1998). The A. humile is a hermaphroditic shrub measuring up to 80 cm (Almeida et al., 1998), with white flowers, diurnal anthesis, a nectar scent, and is pollinated by small insects such as bees and butterflies (Martins and Batalha, 2006). However, there are no detailed studies on its reproductive system. The cashew of A. humile has similar features and use of the related species cashed nut Anacardium occidentale, the only species of cashew tree cultivated in tropics. As with the A. humile, other species of the genus are native and exploited by extractivism. The pseudo fruit of A. humile has an acidic flavor and juicy white pulp and is consumed in natura or in the form of juices, sweets, and jams. Nearly all parts of the plant are used in folk medicine (Almeida et al., 1998; Vieira et al., 2006; Porto et al., 2008). A. humile is a heliophile with a subterraneous stem that can stretch nearly 20 m. The stem stores water, allows the species to withstand long droughts and protects it from fires. Thus, the species has features that make it resistant to the particular environmental water stresses of the Cerrado (Almeida et al., 1998). Study areas Eight natural A. humile populations were selected from the Cerrado biome in the North of Minas Gerais State (Figure 1). Two populations (BAL and CAT) were in a conservation unit (APA Pandeiros) (IEF - Instituto Estadual de Florestas, 2011). The other six populations were from diversified areas and private properties that predominantly had Cerrado vegetation, with some areas that had pastures and had undergone native flora lumber extraction, while others were in a better state of conservation (Table 1). The physiognomic features of Cerrado biome were classified according to Bitencourt et al. (1997). The method proposed by Santos and Vieira (2005) was used to assess the conservation state of the sampling areas, which involved assessing the presence of livestock, fire, and selective cutting to assign a score from 1 to 5 for each area (Table 1). Sampling, DNA extraction, and microsatellite analysis A total of 242 A. humile adult plants were collected from the eight populations and georeferenced (Table 1). The minimum distance between plants collected within a population set was approximately 50 m, given that the stem is subterraneous and could stretch up to 20 m in diameter. Young leaves were collected from each plant for DNA extraction following the protocol proposed by Doyle and Doyle (1987). Genetics and Molecular Research 16 (3): gmr16039778 L.G. Cota et al. 4 Figure 1. Geographic distribution of the Anacardium humile populations in North of Minas Gerais State. For population names, see Table 1. Table 1. Geographical location and sampling details of the Anacardium humile populations studied. Populations Population code Coordinates Sample size Cerrado physiognomies PS Montalvânia MON 14°29’'899''S 44°33'106''W 31 Open cerrado 3 Coração de Jesus COJ 16°34'128''S 44°24'466''W 30 Cerrado sensu stricto 5 Montes Claros MOC 16°50'615''S 43°55'531''W 30 Open cerrado 2 Januária JAN 15°26'493''S 44°28'434''W 30 Closed cerrado 3 APA Pandeiros BAL 15°30'871''S 44°45'176''W 30 Closed cerrado 5 APA Pandeiros CAT 15°17'336''S 44°49'457''W 31 Closed cerrado 5 Claro dos Poções CLP 16°59'395''S 44°04'198''W 30 Open cerrado 2 Campo Azul CAA 16°30'271''S 44°46'113''W 30 Closed cerrado 3 Total 242 PS = preservation status of the area (method proposed by Santos and Vieira, 2005). Six microsatellite loci previously developed for A. occidentale (Croxford et al., 2006) and transferred to A. humile (Cota et al., 2012) were used in population genetic analyses (Table 2). The 15-µL reactions, containing 1X buffer (10 mM Tris-HCl, pH 8.4, 50 mM KCl), 0.7 μM of each primer, 250 μM of each dNTP, 1 U Taq DNA polymerase, 0.25 mg BSA, 1.0 to 1.6 mM MgCl2, 1.25 to 3% formamide, and 9 ng DNA were PCR-amplified using a Veriti thermocycler (Applied Biosystems).

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